Hydroxy-succinimide ester compounds

ABSTRACT

New hydroxy-succinimide ester compounds of the formula ##STR1## wherein X is selected from a carboxy-succinimide ester group and radicals of the general formulae: ##STR2## A is --O--CH 2  --CH 2  when n is 2 and X is a succinimide ester group but otherwise is a single bond; 
     N is a whole number of from 2 to 7; 
     M is 1, 2, 3 or 4, 
     R is hydrogen, methyl, or cyanide; and 
     R&#39; is methyl or ethyl; 
     Have been found outstandingly useful as bridge-building compounds for linking proteins, especially biologically active proteins, to solid or liquid carrier matrix materials, thereby yielding valuable carrier-bound enzymatically active protein compositions.

The present invention relates to new hydroxy-succinimide ester compoundsand to the preparation thereof. An additional aspect of the inventionrelates to the use of such compounds for the production of covalentbondings of proteins, especially of biologically active proteins, toprepare cross-linking or carrier fixed protein compositions.

The interest in immobilized, and especially in carrier-bound,enzymatically active proteins is continuously increasing. Severalprocesses for the covalent bonding of proteins with carrier substancesare already known, for example from German Patent Specifications Nos.2,128,743 and 2,260,185. Immobilized enzymes are particularly importantbecause they result in the stabilization of biologically activematerials and make possible their separation from aqueous reactionmedia.

In the first processes for the covalent bonding of biologically activeproteins, activating groups were introduced into the carrier matrix. Animportant disadvantage of these known methods is the low activity yield,which was attributed to the adverse effect of the carrier matrix on theactive configuration of the protein, due to the close proximity thereof.Attempts were, therefore, made to overcome this disadvantage byintroducing bridge members between the protein and the carrier.

These bridge members are derived from so-called bridge buildingcompounds, which are usually homo- or hetero-difunctional compoundshaving a function which reacts with the protein in aqueous solution,leading to the formation of a covalent bond, and having a furtherfunction capable of forming a bond with the carrier. In the case ofhomodifunctional bridge builders, such as glutardialdehyde ordiepoxides, the two functional groups are the same.

Therefore, protein and carrier are reacted simultaneously, there takingplace not only a protein cross-linking but also a bonding with thecarrier at the same time. Processes have proved to be favorable in whicha two-stage method of working is used, with bonding of the bridgebuilding molecule first with the protein or with the carrier andsubsequently with the carrier or with the protein, respectively. Forthese processes, it has proved to be preferable to useheterodifunctional bridge building compounds with two differentfunctional groups of different reactivity.

Furthermore, it is known first to adsorb the proteins physically on thesurface of appropriate carriers and then to fix them on these surfacesby cross-linking with polyfunctional bridge builders.

In all of these known processes, it has been found that different bridgebuilding compounds behave in a different manner, depending upon theprotein to be bound and upon the carrier material to be used, greatlyvarying results being obtained. Therefore, in practice, for eachparticular case of protein immobilization, the most favorable bridgebuilding substance has to be found. Especially good bridge builders forsome cases have thereby proved to be completely unsatisfactory for othercases. In many cases, a satisfactory covalent bonding of active proteinshas hitherto been completely unsuccessful.

Thus, there is still a need for further bridge building compounds whichprovide a further possibility for the covalent bonding of biologicallyactive proteins and especially of enzymes. In particular, there is aneed for bridge building compounds which can be homodifunctional orheterodifunctional and which contain functional groups reactingespecially quickly and gently with proteins, the distance between thefunctional groups being so great that conformation changes of the boundprotein due to the carrier are substantially excluded and in which the"bridge" which joins the two functional groups can, if desired, bevaried with regard to its hydrophobic or hydrophilic properties. It isan object of the present invention to provide such compounds.

Thus, according to the present invention, there are providedhydroxy-succinimide ester compounds of the formula ##STR3## wherein X isa further carboxy-succinimide ester radical or a radical of the formula##STR4## A is a --O--CH₂ --CH₂ -- radical when n is 2 and X is a furthercarboxy-succinimide ester group but otherwise is a single bond, n is awhole number of from 3 to 7, m is 1, 2, 3 or 4, R is a hydrogen atom ora methyl or cyanide radical and R' a methyl or ethyl radical.

Depending upon the nature of the residue X, the new compounds (I) areeither homodifunctional or heterodifunctional compounds. By variation ofm and n, the hydrophobic or hydrophilic properties of the bridge can be"tailor made", the hydrophobic properties increasing when n is greaterand the hydrophilic properties increasing when m is greater. Ifcross-linking of the proteins is desired, the homodifunctional compoundsof the present invention are preferred, whereas when a bonding with asolid or liquid carrier material is desired, the heterodifunctionalcompounds are preferred in which X is a radical of the formulae (II),(II) or (IV). If bonding with the carrier takes place bycopolymerization, then compounds are especially preferred in which X isa radical of the general formula (III). The hydroxy-succinimide estergroup (HOSu residue) reacts selectively in aqueous solution with aminesand is, therefore, especially useful for the covalent bonding ofproteins.

According to the present invention, the new compounds of general formula(I) can be prepared from the mono- or dicarboxylic acid upon which thehydroxy-succinimide esters are based by either

(a) converting the acid in known manner into a reactive derivative, suchas an acid chloride or mixed anhydride, and reacting the reactivederivative in an organic solvent with N-hydroxy-succinimide, or

(b) condensing the acid directly, in a polar organic organic solvent,with N-hydroxy-succinimide in the presence of an amount ofcyclohexylcarbodiimide equivalent to the latter, or

(c) reacting the acid in the form of an alkali metal salt withN-hydroxy-succinimide-methyl sulphonate in the presence of a crowncompound as catalyst.

The preparation of the compounds (I) via a mixed anhydride in organicsolvent, for example methylene chloride, furan, dioxan or the like,proves, in many cases, to be the simplest and best method. The reactiveacid derivative used is preferably a mixed anhydride with chloroformicacid.

Preferred carrier substances for use with the new compounds according tothe present invention for the covalent bonding of proteins include notonly water-soluble but also water-insoluble, solid or liquid substanceswhich are able to react with the functional groups represented by X, thereactive groups thereby preferably being hydroxy or amino groups.

The carrier substances preferably used include those which arehydrophilic, easily swellable and substantially charge-free, as well asstable towards microorganisms.

According to a preferred process, the compounds according to the presentinvention, when X is a radical of the general formula (III), are bondedby copolymerization with a carrier which is prepared in situ bypolymerization of appropriate monomers or monomer mixtures or areincorporated directly into a carrier by copolymerization withappropriate comonomers.

The copolymerizable monomer used in this case can be water-soluble orwater-insoluble. Water-soluble monomers are preferred when the couplingcompound according to the present invention has first been reacted witha biologically active protein and is thereafter to be fixed on to acarrier in homogeneous aqueous solution, which carrier has been formedin situ by copolymerization. Instead of this, it is also possible to usesuspension or emulsion polymerization methods in which the couplingproduct of a protein with a compound of the general formula (I)according to the present invention is present in the aqueous dispersedphase and a water-insoluble monomer or monomer mixture represents thedispersed phase. Especially preferred comonomers include water-solubleand water-insoluble derivatives of acrylic acid and methacrylic acid,for example the amides, nitriles and esters of these compounds. Amongthe water-soluble comonomers, acrylamide is preferred. Water-insolublederivatives of acrylic acid or methacrylic acid, especially those whichare substituted by alkyl radicals, are preferred when the carrier-boundprotein is subsequently to be used in an aqueous organic medium ratherthan in a purely aqueous system. Other appropriate monomers are derived,for example, from maleic or fumaric acid. However, the monomers whichcan be used are not limited to those mentioned here by way of example.

As monomers, there can also be employed prepolymers which still containunsaturated, copolymerizable groups.

In order to regulate the physical properties in the case of carriersproduced in this manner by copolymerization, appropriate cross-linkersare preferably added in appropriate amounts. Such cross-linkers includecompounds with at least two copolymerizable groups, for exampleN,N'-methylene-bis-acrylamide and ethylene diacrylate. However,radical-acting cross-linkers, for example certain organic peroxides, canalso be employed.

When carrying out the polymerization in the heterogenous phase, i.e. bysuspension or emulsion polymerization, there can be used, for example,water-insoluble cross-linkers, such as divinylbenzene or ethylenedimethacrylate. Numerous other cross-linkers are known and theappropriate choice in a particular case can readily be made. It is,however, also possible subsequently to introduce the cross-linker and tocarry out the cross-linking after the preparation of the polymericcarrier and the bonding with the active protein has taken place.

If the polymer formed is not cross-linked, then soluble or thermoplasticmaterials are obtained. These can be used for spinnable or extrudablesolutions from which the carrier bound proteins can be obtained in knownmanner, for example in the form of filaments or foils.

For coupling with hydroxyl group-containing carrier substances, it isespecially preferred to use those compounds of general formula (I) inwhich X is a radical of general formula (IV). Thus, this radical forms,upon heating to about 100° C. in an organic solvent, for exampledimethyl sulphoxide, by splitting off diethylamine, the correspondingisocyanate of the formula:

    --N═C═O                                            (IVa)

which reacts at 0° C. with hydroxyl groups. Under these conditions, thehydroxy-succinimide ester group (OSu ester) does not react so that aselective reaction of the two functional groups of the molecule ispossible by reaction under the conditions appropriate in each case.

The compounds of general formula (I), in which X is a radical of generalformula (II), are first reacted via the HOSu radical with an aminogroup-containing low or high molecular weight substance and subsequentlyfurther reacted in a weakly acidic aqueous solution. Under theseconditions, from the acetal there is formed the free aldehyde which thenfurther reacts in known manner with, for example, amino groups with theformation of a Schiffs base which is thereafter reduced with, forexample, sodium borohydride, to form a covalent bond. In an analogousmanner, there can also be used hydroxylamine, hydrazine or asemicarbazide derivative.

Furthermore, as carriers there can also be used those solid substanceswhich have no positions reactive with the functional groups of thecompounds according to the present invention. In such cases, thecompounds according to the present invention are used as cross-linkers.The active protein is hereby preferably first applied to the surface ofthe carrier body, for example by adsorption, coating with a solution ofthe protein or the like, followed by anchoring on to this surface bycross-linking with a compound according to the present invention, thehomodifunctional derivatives preferably being used, i.e. those compoundsin which X is a further carboxy-succinimide ester radical. However,compounds in which X is a radical of general formula (IV) can also beused for the cross-linking.

In a further preferred method of using the new compounds according tothe present invention, a biologically active protein is reacted not onlywith a homodifunctional but also with a heterodifunctional compound ofgeneral formula (I). For example, a protein is first reacted with acompound in which X is a radical of general formula (III), which leadsto the introduction of copolymerizable residues. Subsequently, across-linkable and preferably a homodifunctional compound is addedthereto in which X is a further carboxysuccinimide ester group. Thisleads to a cross-linking, several molecules thus being linked with oneanother. This complex body, consisting of several protein units, canthen, as described above, be immobilized on a carrier by means of thecopolymerizable residues present therein, according to theabove-described methods of protein copolymerization. In the same way, itis also possible, instead of a copolymerizable group, to introduce aradical of the general formula (II) or (IV) according to this method andto produce the bonding with a preformed carrier.

According to a further variant, the protein is first cross-linked, withthe bonding together of several protein molecules, followed byimmobilization on a carrier with the same or with a different compoundof general formula (I).

Biologically active proteins which can be covalently bound with thecompounds according to the present invention include enzymaticallyactive proteins and immunologically active proteins, such as antibodies,immunoglobins and the like, as well as hormone-active proteins.

The new compounds according to the present invention are characterizedby their reactivity with proteins which, in aqueous or aqueous-organicsolution, leads to the formation of covalent bonds with the substantialmaintenance of the biological activity, as well as by their reactivitywith copolymerizable double bonds, hydroxyl groups and amino groups andalso with other active groups present in the carrier substances forproteins. A further advantage of these new esters is that theycrystallize well and can, therefore, easily be isolated in pure form.They can form covalent bondings with carriers under conditions in whichthe biological activity of the proteins remains intact. Furthermore,their hydrophilic or hydrophobic properties can easily be varied, asdesired. Thus, for example, symmetrical bis-hydroxy-succinimide esterscan be added in the particular appropriate amount to a solution of aprotein in a weakly alkaline buffer. The cross-linked biologicallyactive proteins thereby obtained can either be separated off, forexample by dialysis, ultrafiltration or the like, or can be separatedoff, for example by gel chromatography, or can be further reacted, forexample by bonding on to a carrier or polymerizing into one. Thecompounds according to the present invention derived from ethyleneglycol are thereby superior to simple dicarboxylic acidhydroxy-succinimide esters because of their hydrophilic properties.Because of their better water solubility, they react more quickly anddenaturing of biologically active proteins is suppressed.

In the case of the asymmetrical (heterodifunctional) compounds, it is,for example, possible to convert the amido-acetaldehyde-acetal group,after coupling of the hydroxy-succinimide group with a protein, into thefree aldehyde by acidification, followed, as described above, byreaction with a further amino group-containing substance.

Therefore, it is possible to couple, for example, not only two differentenzymes with one another, but also an enzyme on to albumin or an antigenon to bovine serum albumin or on to a polyamino acid, such as polyamine.

The compounds containing a copolymerizable double bond can be used, forexample, according to the methods described in German PatentSpecifications Nos. 2,130,913 and 2,128,743, for proteincopolymerization. It is also possible first to subject these compoundsto a copolymerization, whereby, depending upon the choice of thecomonomers, readily or less-readily water-soluble copolymers areobtained. Proteins can then be coupled on to these copolymers, which canbe prepared with any desired molecular weight, via thehydroxy-succinimide ester group.

The following Examples are given for the purpose of illustrating thepresent invention:

EXAMPLE 1 Ethylene glycol-bis-propionic acid bis-hydroxy-succinimideester

To a solution of 10.3 g. ethylene glycol bis-propionic acid (preparedaccording to the method of R. V. Christian and R. M. Hixon, J.A.C.S.,70, 1333/1948; Doc. No. 12916) and of 12.6 g. N-hydroxy-succinimide in150 ml. anhydrous tetrahydrofuran, there is added dropwise, withstirring and ice cooling, over the course of one hour, a solution of22.7 g. dicyclhexyl carbodiimide in tetrahydrofuran. The solution isstirred for 18 hours at ambient temperature. 0.3 ml. acetic acid arethen added thereto. After a further hour, 150 ml. anhydrous ethylacetate are added thereto and precipitated dicyclohexylurea is filteredoff. The filtrate is concentrated to one third, washed with water,aqueous sodium bicarbonate solution and again with water, dried overanhydrous sodium sulphate and evaporated. The residue is taken up inacetonitrile, left to stand for 24 hours at +4° C., then filtered offfrom freshly precipitated urea and again evaporated. After standing forseveral days at +4° C., the desired product thus obtained crystallizesout from the residual oil. It is recrystallized from ethyl acetate;yield: 25% of theory; m.p. 115° C.

Analysis: C₁₆ H₂₀ N₂ O₁₀ (M.W. 400.35) calculated: C, 47.99%; H, 5.04%;N, 6.99%. Found: C, 47.95%; H, 5.04%; N, 6.87%.

EXAMPLE 2

In a manner analogous to that described in Example 1, there is preparedoxy-bis-propionic acid hydroxysuccinimide ester; yield: 30% of theory;m.p.: 131° C.

EXAMPLE 3 Oxy-succinimide-glutaric acid aminoacetaldehyde dimethylacetal

To a solution of 11.4 g. glutaric acid anhydride in 100 ml.tetrahydrofuran there is simultaneously dropped in 10.1 g. triethylamineand 13.2 g. aminoacetaldehyde dimethylacetal, whereafter the reactionmixture is stirred for 30 minutes. The resultant solution is cooled to+5° C. and slowly mixed with 9.5 ml. ethyl chloroformate. The reactionmixture is then kept for 30 minutes at -5° C. Subsequently, 11.5 g.hydroxysuccinimide and 14.0 ml. triethylamine are simultaneously addeddropwise at 0° C. After reaction for 5 hours at +25° C., precipitatedtriethyl ammonium chloride is filtered off, the filtrate is evaporatedand the residue is again taken up in ethyl acetate, washed with water,aqueous sodium bicarbonate solution and again with water, dried overanhydrous sodium sulphate and evaporated. The desired product thusobtained is recrystallized from hot ethyl acetate; yield: 45% of theory;m.p.: 70° C.

Analysis: C₁₃ H₂₀ N₂ O₇ (M.W. 316.3) calculated: C, 49.60%; H, 6.38%; N,8.85%. Found: C, 49.34%; H, 6.37%; N, 8.81%.

EXAMPLE 4 Methacryloxyl-ω-hydroxy-carboxylic acid hydroxysuccinimideester

The methacryloxyl-ω-hydroxycarboxylic acids are prepared in a manneranalogous to the process described in Makromol. Chem., 176, 3017/1973).In a 500 ml. three-necked flask are placed 0.1 mol of themethacryloylhydroxy acid in question and 0.11 mol N-hydroxysuccinimidein a mixture of anhydrous methylene chloride and anhydrous methylenechloride and anhydrous tetrahydrofuran and then cooled in an ice-bath to+5° C. A solution of 0.11 mol dicyclohexylcarbodiimide, dissolved in 50ml. anhydrous methylene chloride, is added dropwise thereto, whilestirring. The reaction mixture is further stirred for about 12 hours,the temperature thereby increasing to +25° C. Precipitateddicyclohexylurea is thereafter filtered off and the filtrate isevaporated. The resulting oil is taken up in acetonitrile and left tostand for several hours in a refrigerator. Further precipitated urea isfiltered off and the acetonitrile is evaporated off from the filtrate.The product is thus obtained in the form of an oil.

EXAMPLE 5 Hydroxysuccinimido-N-2-hydroxyethyl-N-',N'-dimethylureasuccinic acid ester

13.2 g. N-2-hydroxyethyl-N',N'-dimethylurea are dissolved in anhydroustetrahydrofuran. 2.4 g. of a sodium hydride suspension are then addeddropwise in the course of about 5 minutes. The reaction mixture isstirred at ambient temperature until the evolution of hydrogen hasceased, whereafter 10 g. succinic anhydride are added thereto. Afterstirring for a further 24 hours, the reaction mixture is evaporated andwater is added thereto. After the evolution of hydrogen has ceased, theorganic components are separated off and the solution is acidified andshaken out with methylene chloride. The organic phase is dried andevaporated, a pale yellow oil remaining behind; yield: 53% of theory.

The hydroxysuccinimide ester was prepared from this acid in a manneranalogous to that described in Example 4; yield: 35% of theory; m.p.:97°-98° C.

Analysis: calc.: C, 47.5%; H, 5.8%; C, 12.8%. Found: C, 47.5%; H, 5.8%;C, 12.8%.

By heating to about 100° C. in dimethyl sulphoxide, diethylamine issplit off with the formation of the corresponding isocyanate.

The following Examples illustrate the use of the new compounds accordingto the present invention:

EXAMPLE 6

Stabilization of trypsin by cross-linking with various symmetricalbifunctional compounds.

Comparative experiment.

In a first experiment, trypsin was reacted with glutaraldehyde and, in asecond experiment, with suberic acid bis-imido ester hydrochloride, thedecrease of activity of the thus cross-linked trypsin being comparedwith that of free trypsin. To 50 mg. amounts of trypsin in 25 ml.distilled water, there were added 2 μM of the cross-linker substance,dissolved in 0.05 M phosphate buffer (pH 7.5), followed by stirring atambient temperature. After 20 hours, the activity of the free trypsin,as well as of the trypsin cross-linked with glutaraldehyde, had droppedto 15% of the initial activity. After 50 hours, the activity in allthree samples had dropped to 0. The trypsin cross-linked with subericacid bis-imido ester hydrochloride still had, after 50 hours, 50%, after50 hours still 30% and after 100 hours still 20% of the initialactivity.

The experiment was repeated with the esters of Examples 2, 3 and 4according to the present invention. After 20 hours, the activity withthe compound of Example 3 was still 85% and with that of Example 4 still50%. After 50 hours, the activity of the trypsin cross-linked with thecompound of Example 2 was 43% and with the compound of Example 3 was27%. After 100 hours, with the compound of Example 2, an activity of 25%was measured. This Example 6 shows the superiority of the compoundsaccording to the present invention, in comparison with knowncross-linking agents.

EXAMPLE 7

Stabilization of kidney acylase by cross-linking.

The activity of kidney acylase in 0.1 M phosphate buffer (pH 4) drops,in spite of cooling to 4° C., within 3 to 5 days to 0. If, however, thecompound of Example 1 or suberic acid bis-hydroxysuccinimide ester(prepared analogously to Example 1) is added thereto, then an initialdrop of activity takes place to 70 to 50%. This remaining activity wasmonitored for 2 months and remained constant over this period of time.This result is always achieved in the case of variation of the mol ratioof protein to ester of from 1:1 to 1:8.

A comparative experiment with glutaraldehyde instead of the bis-esteraccording to the present invention does not lead to any stabilization.

EXAMPLE 8

Immobilization of kidney acylase by copolymerization.

1000 mg. aminoacylase (lyophilisate) (specific activity 17.0 U/mg.) aredissolved in 25.0 ml. 1 M tris buffer (pH 8.3) at +4° C. To this areadded 2.5 ml. of a solution of 30.7 mg. methacrylic hydroxycapronic acidhydroxysuccinimide ester (see Example 4) in dioxan. The reaction time is12 hours at +4° C.

6 g. Acrylamide, 0.5 g. N,N'-methylene-bis-acrylamide and 0.6 ml. of a1% cobalt chloride solution are dissolved in 22.5 ml. of the above trisbuffer. After the addition of the vinylated enzyme solution,polymerization is started with 1.5 ml. each of a 5% ammoniumperoxydisulphate solution and of a 5% 3-dimethylaminopropionitrilesolution. The resultant gel is pressed through a sieve (mesh size 0.4mm.) and eluted in a column with 1 M phosphate buffer (pH 7.5).

1st eluate: 2 liters buffer 1.88% activity in eluate; specific activityon the gel 193.1 U/g.

2nd eluate: 2 liters buffer-- specific activity on the gel 193.1 U/g.

Activity yield: 6.9%.

The Example was repeated with the use of acryloyl chloride instead ofthe compound according to the present invention: no activity was boundon to the carrier.

EXAMPLE 9

Immobilization of glucose oxidase (GOD) by a 2 stage reaction withcompounds according to the present invention and subsequentcopolymerization.

As described in Example 8, GOD with a specific activity of about 210U/mg. was pre-incubated with the compound of Example 4 and subsequentlya small amount of the compound of Example 1 was added thereto.Thereafter, the reaction mixture was left to stand for a further 24hours and subsequently copolymerization was carried out in the mannerdescribed in Example 8. An activity of 290 U/g. was measured in the gelobtained.

A repetition of this experiment with the use of acryloyl chlorideinstead of the compounds according to the present invention gave anactivity of 235 U/g.

EXAMPLE 10

Coupling of angiotensin on to BSA (bovine serum albumin).

25 mg. angiotensin were dissolved in 10 ml. dioxan/water (1:1 v/v) andmixed with a solution of 6 mg. of the compound of Example 4 in dioxan.The pH value was then brought to 8.0 by the addition of 5% aqueouspotassium carbonate solution and stirred for 2 days at ambienttemperature. The product was precipitated out by the addition oftetrahydrofuran, centrifuged off and lyophilised; yield: 19.6 mg. oftheory.

The 19.6 mg. of this intermediate were adjusted to pH 4 and stirred for1 hour. Subsequently, the pH value was again adjusted to 8.0 and thesolution mixed with 16.6 mg. BSA. After stirring for one hour, thereaction mixture was mixed with 1 mg. sodium borohydride. After afurther 20 minutes, a further 1 mg. sodium borohydride was added. Aftera further 60 minutes, the reaction was finished. The solution wasevaporated and the residue was taken up in absolute ethanol, filtered,water added thereto and then dialysed for 4 days. From the dialysatethere were obtained 24.1 mg. lyophilisate. From this it follows that atleast 24 molecules angiotensin are coupled with 1 molecule BSA.

EXAMPLE 11

Copolymerization of the ω-methacrylol-hydroxycarboxylicacid-hydroxysuccinimide esters with methacrylamide.

There was prepared an oxygen-free solution (1 mol/dm³) of the twomonomers in anhydrous tetrahydrofuran (monomer ratio ester:amide=1:5).Polymerization took place by the addition of 0.8% (mol/mol)azoisobutyric acid dinitrile at 58° C. After 4 hours, polymerization wasdiscontinued by cooling (dipping into an ice-bath) and the precipitatedpolymer was filtered off with suction. It was again dissolved in waterand reprecipitated by dropping the aqueous solution into acetone.

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. Hydroxy-succinimide ester compound of the formula##STR5## wherein X is

    --CO--NH--CH.sub.2 --CH(OR').sub.2

A is a single bond; n is a whole number of from 2 to 7; and R' is methylor ethyl.
 2. Hydroxy-succinimide ester compound as claimed in claim 1wherein R' is methyl.
 3. Hydroxy-succinimide ester compound as claimedin claim 1 wherein R' is ethyl.
 4. Hydroxy-succinimide ester compound asclaimed in claim 1 designated oxy-succinimide-glutaric acidaminoacetaldehyde dimethyl acetal.